Plasmodiophora brassicae is a protozoan pathogen that causes clubroot disease in cruciferous plants, particularly Chinese cabbage (Brassica rapa). A previous study identified a clubroot resistance gene (CRd) conferring race-specific resistance to P. brassicae. However, the defense mechanisms of B. rapa against virulent vs. avirulent P. brassicae are poorly understood. In this study, we carried out a global transcriptional analysis in the clubroot-resistant Chinese cabbage inbred line “85–74” carrying the CRd gene and inoculated with avirulent (LAB-4) or virulent (SCCD-52) P. brassicae. RNA sequencing showed that “85–74” responded most rapidly to SCCD-52 infection, and the number of differentially expressed genes was much higher in SCCD-52-treated as compared to LAB-4-treated plants (5552 vs. 304). Transcriptome profiling revealed that plant hormone signal transduction and plant–pathogen interaction pathways played key roles in the late stages of P. brassicae infection. Genes relating to the salicyclic acid (SA), jasmonic acid (JA)/ethylene (ET), and brassinosteroid (BR) signaling pathways were up-regulated relative to untreated plants in response to LAB-4 infection at 8, 16, and 32 days post-inoculation (dpi) whereas JA, ET, and BR signaling-related genes were not activated in response to SCCD-52, and SA signaling-related genes were up-regulated in both LAB-4 and SCCD-52, suggesting that SA signaling is not the key factor in host resistance to avirulent P. brassicae. In addition, genes associated with phosphorylation and Ca2+ signaling pathways were down-regulated to a greater degree following LAB-4 as compared to SCCD-52 infection at 8 dpi. These results indicate that effector-triggered immunity in “85–74” is more potently activated in response to infection with avirulent P. brassicae and that JA, ET, and BR signaling are important for the host response at the late stage of infection. These findings provide insight into P. brassicae pathotype-specific defense mechanisms in cruciferous crops.

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Background: Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.Results: In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without a pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5 to 4.0 months. Average transformation frequency is up to 10.83%. Furthermore, using this transformation system, the CRISPR/Cas9 technology was successfully applied in Chinese cabbage by knocking out a self-incompatibility-related gene SRK. Gene sequencing analysis in the positive transgenic lines revealed various mutations, including deletions, insertions, and substitutions. Conclusion: A simple, stable and efficient genetic transformation method was established for Chinese cabbage and successfully applied to the CRISPR/Cas9 system. The results of this study pave the way for further gene functional studies and genome editing in Chinese cabbage.